Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these ␤-glucans have immunomodulating properties. We show that the overproduction of the Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter ؊1 ) of a position 2-substituted (133)-␤-D-glucan that has potential use as a food additive.

To define the active site of the 5-3 exonucleolytic domain of the Streptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His-tagged Spn pol I fusion protein and introduced mutations at residues Asp 10 , Glu 88 , and Glu 114 , which are conserved among all prokaryotic and eukaryotic 5 nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K mutants and the control fusion protein support: (i) a critical function of Asp 10 in the catalytic event, (ii) a role of Glu 114 in the exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu 88 for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5 nucleases.The polymerase function of type-I-like DNA polymerases has been studied in considerable detail, with biochemical and mutagenesis analysis proceeding in parallel with the structural studies (1-6). Such detailed study have provided a prototypical molecular model of DNA-dependent DNA polymerization and important insights into the architecture of the primer and nucleotide binding sites. By contrast, despite the mutational and structural analysis of several 5Ј-3Ј exonucleolytic domains of eubacterial polymerases (7-9) and related bacteriophage 5Ј nucleases (10 -12), the molecular mechanism of the exonucleolytic reaction still remains obscure.Sequence comparisons and enzymatic studies indicate that the eubacterial pol 1 I-associated 5Ј nucleases share significant sequence homology with the polymerase-independent 5Ј nucleases from several bacteriophages (13). The prokaryotic 5Ј nucleases are also related to mammalian FEN-1 proteins and several yeast proteins of the RAD2 family, having two large blocks of sequence similarity that bear some resemblance to the bacterial and bacteriophage nuclease sequences (14, 15). Some clues to identify important residues in the bacterial 5Ј-3Ј exonuclease family derive from the multiple sequence alignment of 10 bacterial and bacteriophage nucleases (13). Six conserved sequence motifs containing 14 invariant amino acids were identified, 9 of which were carboxylate residues. The presence of highly conserved carboxylate residues led to the proposal that some of these amino acids could be involved in metal binding at the active site of the 5Ј-3Ј exonucleases, as occurs in other enzymes catalyzing phosphoryl transfer reactions (1). This hypothesis has been further supported by structural data from the 5Ј nucleases from bacteriop...

-Citrate metabolism performed by a number of lactic acid bacteria yields volatile compounds, such as diacetyl and acetaldehyde, which are important for flavour development in fermented milk products. Citrate uptake in Lactococcus laetis subsp. laetis biovar diacetylactis and Leuconostoc mesenteroides subsp. mesenteroides is catalyzed by a secondary carrier, the citrate permease P (CitP). The presence of CitP is essential for citrate utilization, since in its absence no citrate metabolism is observed although ail enzymes involved in conversion of citrate are present inside the cells. In this review the genetic organization of the plasmid encoding the citrate transport system of lactococci is described, the posttranscriptional regulation of the citQRP operon is presented and the influence of external pH on citP transcription, citrate uptake and cometabolism of citrate and glucose is discussed. These last studies reveal a novel molecular mechanism that improves the adaptation of L. diacetylactis to acidic pH. © Inra/Elsevier, Paris.Lactococcus lactis subsp. lactis biovar diacetylactis / citrate transport 1 regulation of gene expression 1 lactic acid bacteria 1 acidic stress Résumé -Régulation de 1'expression du système de transport du citrate chez Lactococcus lactis subsp.lactis biovar diacetylactis. Le métabolisme du citrate chez certaines bactéries lactiques se traduit par la synthèse de composés d'arôme tels que le di acétyle et l'acétaldéhyde. Ces composés contribuent à l'élaboration de la flaveur de certains aliments issus de la fermentation lactique. Le transport du citrate chez Lactococcus lactis subsp. lactis biovar diacetylactis et Leuconostoc mesenteroides subsp mesenteroides est catalysé par un transporteur secondaire, la citrate perméase P. Cette perméase est essentielle pour l'utilisation du citrate. En son absence le citrate n'est pas métabolisé, bien que toutes les enzymes impliquées dans sa conversion soient pré-sentes au niveau de la cellule. Dans cette revue, nous décrivons l'organisation génétique du plasmide codant pour le transport du citrate chez L. diacetylactis, la régulation post-transcriptionnelle de l'opéron citQRP et l'influence du pH extracellulaire sur la transcription du gène citP. Par ailleurs, l'étude du transport du citrate et du cométabolisme citrate-glucose a révélé un nouveau mécanisme moléculaire permettant une meilleure adaptation de L. diacetylaetis à des pH acides. © InralElsevier, Paris.Lactococcus lactis subsp.lactis biovar diacetylactis 1 transport du citrate! génétique! bactérie lactique! stress acide

The citMCDEFGRP cluster from Leuconostoc paramesenteroides involved in citrate utilization was cloned and its nucleotide sequence determined. Homology of the inferred gene products with characterized enzymes reveals that citP encodes the citrate permease P, citC the citrate ligase and citDEF the subunits of the citrate lyase of Leuconostoc. Moreover, it suggests that citM encodes a Leuconostoc malic enzyme. Analysis of citrate consumption by and citrate lyase activity of Lc. paramesenteroides J1[pCITJ1] showed that its citrate permease and its citrate lyase are induced by the presence of citrate in the growth medium. Southern blot analysis demonstrated that the citMCDEFGRP cluster is located in a plasmid.